Immunogenicity of chimeric hemagglutinins delivered by an orf virus vector platform against swine influenza virus.

Orf virus (ORFV) is a large DNA virus that can harbor and efficiently deliver viral antigens in swine. Here we used ORFV as a vector platform to deliver chimeric hemagglutinins (HA) of Influenza A virus of swine (IAV-S). Vaccine development against IAV-S faces limitations posed by strain-specific immunity and the antigenic diversity of the IAV-S strains circulating in the field. A promising alternative aiming at re-directing immune responses on conserved epitopes of the stalk segment of the hemagglutinin (HA2) has recently emerged. Sequential immunization with chimeric HAs comprising the same stalk but distinct exotic head domains can potentially induce cross-reactive immune responses against conserved epitopes of the HA2 while breaking the immunodominance of the head domain (HA1). Here, we generated two recombinant ORFVs expressing chimeric HAs encoding the stalk region of a contemporary H1N1 IAV-S strain and exotic heads derived from either H6 or H8 subtypes, ORFVΔ121cH6/1 and ORFVΔ121cH8/1, respectively. The resulting recombinant viruses were able to express the heterologous protein in vitro. Further, the immunogenicity and cross-protection of these vaccine candidates were assessed in swine after sequential intramuscular immunization with OV-cH6/1 and OV-cH8/1, and subsequent challenge with divergent IAV-S strains. Humoral responses showed that vaccinated piglets presented increasing IgG responses in sera. Additionally, cross-reactive IgG and IgA antibody responses elicited by immunization were detected in sera and bronchoalveolar lavage (BAL), respectively, by ELISA against different viral clades and a diverse range of contemporary H1N1 IAV-S strains, indicating induction of humoral and mucosal immunity in vaccinated animals. Importantly, viral shedding was reduced in nasal swabs from vaccinated piglets after intranasal challenge with either Oh07 (gamma clade) or Ca09 (npdm clade) IAV-S strains. These results demonstrated the efficiency of ORFV-based vectors in delivering chimeric IAV-S HA-based vaccine candidates and underline the potential use of chimeric-HAs for prevention and control of influenza in swine.

A Novel Recombinant Newcastle Disease Vaccine Improves Post- In Ovo Vaccination Survival with Sustained Protection against Virulent Challenge.

In ovo vaccination has been employed by the poultry industry for over 20 years to control numerous avian diseases. Unfortunately, in ovo live vaccines against Newcastle disease have significant limitations, including high embryo mortality and the inability to induce full protection during the first two weeks of life. In this study, a recombinant live attenuated Newcastle disease virus vaccine containing the antisense sequence of chicken interleukin 4 (IL-4), rZJ1*L-IL4R, was used. The rZJ1*L-IL4R vaccine was administered in ovo to naïve specific pathogen free embryonated chicken eggs (ECEs) and evaluated against a homologous challenge. Controls included a live attenuated recombinant genotype VII vaccine based on the virus ZJ1 (rZJ1*L) backbone, the LaSota vaccine and diluent alone. In the first of two experiments, ECEs were vaccinated at 18 days of embryonation (DOE) with either 104.5 or 103.5 50% embryo infectious dose (EID50/egg) and chickens were challenged at 21 days post-hatch (DPH). In the second experiment, 103.5 EID50/egg of each vaccine was administered at 19 DOE, and chickens were challenged at 14 DPH. Chickens vaccinated with 103.5 EID50/egg of rZJ1*L-IL4R had hatch rates comparable to the group that received diluent alone, whereas other groups had significantly lower hatch rates. All vaccinated chickens survived challenge without displaying clinical disease, had protective hemagglutination inhibition titers, and shed comparable levels of challenge virus. The recombinant rZJ1*L-IL4R vaccine yielded lower post-vaccination mortality rates compared with the other in ovo NDV live vaccine candidates as well as provided strong protection post-challenge.

Newcastle Disease Virus Induced Pathologies Severely Affect the Exocrine and Endocrine Functions of the Pancreas in Chickens.

Newcastle disease virus (NDV) causes a highly contagious and devastating disease in poultry. ND causes heavy economic losses to the global poultry industry by decreasing the growth rate, decrease in egg production high morbidity and mortality. Although significant advances have been made in the vaccine development, outbreaks are reported in vaccinated birds. In this study, we report the damage caused by NDV infection in the pancreatic tissues of vaccinated and specific-pathogen-free chickens. The histopathological examination of the pancreas showed severe damage in the form of partial depletion of zymogen granules, acinar cell vacuolization, necrosis, apoptosis, congestion in the large and small vessels, sloughing of epithelial cells of the pancreatic duct, and mild perivascular edema. Increased plasma levels of corticosterone and somatostatin were observed in NDV-infected chicken at three- and five- days post infection (DPI). A slight decrease in the plasma concentrations of insulin was noticed at 5 DPI. Significant changes were not observed in the plasma levels of glucagon. Furthermore, NDV infection decreased the activity and mRNA expression of amylase, lipase, and trypsin from the pancreas. Taken together, our findings highlight that NDV induces extensive tissue damage in the pancreas, decreases the activity and expression of pancreatic enzymes, and increases plasma corticosterone and somatostatin. These findings provide new insights that a defective pancreas may be one of the reasons for decreased growth performance after NDV infection in chickens.

Runting and Stunting Syndrome in Broiler Chickens: Histopathology and Association With a Novel Picornavirus.

Runting stunting syndrome (RSS) in commercial chickens has been reported worldwide, and although several studies have attempted to clarify the cause and describe the lesions, there are gaps in knowledge of the epidemiology, pathogenesis, and etiology. The study objective was to use commercial chicks naturally affected by RSS to describe the histologic changes of RSS in all segments of the small intestine in chicks of different ages and to identify viral gene sequences in affected chicks and their association with histologic lesions. Chicks lacking clinical signs but from the same houses and from unaffected houses were used as controls. The average weight of affected chicks was significantly lower than expected for their flocks. Macroscopically, the small intestines had paler serosa, with watery, mucoid, or foamy contents and poorly digested food. Histologic lesions were characterized by necrotic crypts, crypt dilation, and flattening of the crypt epithelium. Histomorphometry of the intestines revealed villous atrophy especially in the jejunum and ileum. Histologic changes in other organs were not observed. Random next-generation sequencing of total RNA extracted from formalin-fixed paraffin-embedded tissues detected avian nephritis virus, avian rotavirus, and picornavirus in jejunal segments from 7-day-old chicks. No viruses were detected in the jejunum of 1-day-old chicks. Detection of picornaviral reads was significantly associated (P < .05) with histologic lesions of RSS. Sequence analysis of the picornavirus revealed genetic similarity with the genus Gallivirus. Using in situ hybridization for galliviral nucleic acid sequences, the signal was associated with crypt lesion severity, although signal was detected both in chicks with and without RSS.

Newcastle disease virus induces testicular damage and disrupts steroidogenesis in specific pathogen free roosters.

Newcastle disease (ND), which is caused by Newcastle disease virus (NDV), can cause heavy economic losses to the poultry industry worldwide. It is characterised by extensive pathologies of the digestive, respiratory, and nervous systems and can cause severe damage to the reproductive system of egg-laying hens. However, it is unknown whether NDV replicates in the male reproductive system of chickens and induces any pathologies. In this study, we selected a representative strain (i.e. ZJ1) of the most common genotype (i.e. VII) of NDV to investigate whether NDV can induce histological, hormonal, and inflammatory responses in the testes of specific pathogen free (SPF) roosters. NDV infection increased the expression of toll like receptor TLR3, TLR7, MDA5, IFN-α, IFN-ß, IFN-γ, IL-8, and CXCLi1 in the testes of NDV-infected roosters at 5 days post-infection (dpi). Severe histological changes, including decrease in the number of Sertoli cells and individualized, shrunken spermatogonia with pyknotic nuclei, were observed at 3 dpi. At 5 dpi, the spermatogenic columns were disorganized, and there were fewer cells, which were replaced by necrotic cells, lipid vacuoles, and proteinaceous homogenous material. A significant decrease in the plasma concentrations of testosterone and luteinizing hormone (LH) and the mRNA expression of their receptors in the testes, steroidogenic acute regulatory protein, cytochrome P450 side-chain cleavage enzyme, and 3ß-hydroxysteroid dehydrogenase in the NDV-infected group was observed relative to those in the control group (P < 0.05). Collectively, these results indicate that NDV infection induces a severe inflammatory response and histological changes, which decrease the steroidogenesis.

Enhanced phylogenetic resolution of Newcastle disease outbreaks using complete viral genome sequences from formalin-fixed paraffin-embedded tissue samples.

Highly virulent Newcastle disease virus (NDV) causes Newcastle disease (ND), which is a threat to poultry production worldwide. Effective disease management requires approaches to accurately determine sources of infection, which involves tracking of closely related viruses. Next-generation sequencing (NGS) has emerged as a research tool for thorough genetic characterization of infectious organisms. Previously formalin-fixed paraffin-embedded (FFPE) tissues have been used to conduct retrospective epidemiological studies of related but genetically distinct viruses. However, this study extends the applicability of NGS for complete genome analysis of viruses from FFPE tissues to track the evolution of closely related viruses. Total RNA was obtained from FFPE spleens, lungs, brains, and small intestines of chickens in 11 poultry flocks during disease outbreaks in Pakistan. The RNA was randomly sequenced on an Illumina MiSeq instrument and the raw data were analyzed using a custom data analysis pipeline that includes de novo assembly. Genomes of virulent NDV were detected in 10/11 birds: eight nearly complete (> 95% coverage of concatenated coding sequence) and two partial genomes. Phylogeny of the NDV complete genome coding sequences was compared to current methods of analysis based on the full and partial fusion genes and determined that the approach provided a better phylogenetic resolution. Two distinct lineages of sub-genotype VIIi NDV were identified to be simultaneously circulating in Pakistani poultry. Non-targeted NGS of total RNA from FFPE tissues coupled with de novo assembly provided a reliable, safe, and affordable method to conduct epidemiological and evolutionary studies to facilitate management of ND in Pakistan.